The phagocytic cells of the peripheral blood mononuclear population are a major source of potent serine proteases that play essential roles in the pathophysiology of tissue homeostasis, inflammation and blood coagulation. The neutral proteases secreted by stimulated monocytes/macrophages include elastase, plasminogen-activators, and collagenases, as well as components of both the coagulation and complement pathways. In addition to contributing to the structural reorganization of tissue, particularly at inflammatory sites, some of these proteases also appear to be instrumental in the control of mononuclear cell and tumor cell migration and both the cytolytic and mitogenic activities of macrophages.
The mechanism universally employed to regulate the destructive effects of serine proteases is the coordinated synthesis of specific endogenous inhibitors. These serine protease inhibitors constitute a family of closely related and structurally highly conserved proteins (the Serpin superfamily [Carrell, R. and Travis, J. (1985) Trends Biol. Sci. 10:20-24]), with the common functional feature that they "trap" the protease by presenting a reactive site that provides an "ideal" pseudo-substrate (Travis, J. and Salvesen G. S. [1983] Ann. Rev. Biochem. 52:655-709). The target specificity of each Serpin (i.e., Met or Val for elastase; Leu for chymase; Arg for thrombin) is determined by the amino acid residue located at its reactive center, which is normally the same amino acid found on the amino-terminal side of the peptide bond to be cleaved by the protease in the legitimate substrate (Travis and Salveson, supra). This crucial amino acid forms the basis for a Serpin classification scheme (e.g., Met-Serpin, Arg-Serpin, etc. [Carrell, R. and Travis, J. (1985), supra]. The enzyme and its inhibitor bind tenaciously to form a complex that is typically stable to boiling in detergent, but is susceptible to neutrophilic cleavage with ammonium hydroxide. Failure or lack of regulation of these powerful proteolytic enzymes is known to lead to severe pathological effects (Carrell and Travis, supra; Carrell, R. W. and Owen, M. C. [1985] Nature 317:730-732). For example, a genetic or environmentally induced deficiency in the most prevalent serum Met-Serpin, .alpha..sub.1 -protease inhibitor (API), has been demonstrated to account for the loss of pulmonary function associated with emphysema (Morse, J. O. [1978] N. Engl. J. Med. 299:1099-1105). Conversely, an overproduction of the Arg-Serpin that inhibits the action of the plasminogen activator (PA) found in plasma, has been correlated with human thromboembolic disease (Hamsten, A., Wiman, B., de Faire, U. and Blomback, M. [1985] N. Eng. J. Med. 313:1557-1563).
An endothelial cell plasminogen activator inhibitor (PAI-1) clone was recently reported (Pannekoek, H., Veerman, H., Lambers, H., Diergaarde, P., Verweij, C. L., van Zonneveld, A.-J. and van Mourik, J. A. [1986] EMBO Jour. 5:2539-2544; Ginsburg, D., Zeheb, R., Yang, A. Y., Rafferty, U. M., Andreasen, P. A., Nielsen, L., Dano, K., Lebo, R. V. and Gelehrter, T. D. [1986] J. Clin. Invest. 78:1673-1680; Ny, T., Sawdey, M., Lawrence, D., Millan, J. L. and Loskutoff, D. J. [1986] Proc. Natl. Acad. Sci. USA 83:6776-6780). This is distinctly different from the monocyte-derived cDNA coding for PAI-2, the subject of this invention.
A recently published (Kruithof, E. K. O., Vassalli, J.-D., Schleuning, W.-D., Mattaliano, R. J. and Bachmann, F. [1986] J. Biol. Chem. 261:11207-11213) partial amino acid sequence (30 carboxyl residues) of monocyte-derived PAI-2 was found to exactly match residues 347-376 (boxed in FIG. 3A) of the amino acid sequence deduced from our clone pcD-1214. Perfect homology over 30 amino acid residues strongly suggests that the monocyte Arg-Serpin identified in this is the same protein isolated and characterized biochemically from U937 cells by Kruithof et al., and now classified as PAI-2 (Kruithof et al., supra). Northern blots of mRNA from stimulated U937 cells probed with the monocyte Arg-Serpin clone confirm transcriptional activity of this gene in these cells (FIG. 2; lane 5).
Monocyte PAI (PAI-2) is synthesized upon stimulation of monocytes/macrophages with lipopolysaccharide (LPS), phorbol ester (PMA), or muramyl dipeptide (MDP). The biochemical properties of the PAI-2 distinguish it from both protease-nexin secreted by fibroblasts (Scott, R. W., Eaton, D. L., Duran, N. and Baker, J. B. [1983] J. Biol. Chem. 258:4397-4403) and the endothelial cellderived PAI-1(Pannekoek et al. supra).